免费文献传递   相关文献

Cloning,expression and characterization of exo-inulinase from Kluyveromyces marxianus DSM 5418

一个外切菊粉酶基因的克隆表达及酶学性质



全 文 :!11
"!

#
2013
$

%
&
 

 
(
 
)
 
*
 
+
ChineseJournalofBioprocessEngineering
Vol.11No.3
May2013
doi:10.3969/j.issn.1672-3678.2013.03.008
!"#$
:2012-02-23
%&(

«’¢ÃŽôV±é56
(2010GXNSFD013030);
«òó:!rŽOœxç1j5ìV

«ÃŽÄÄôV
(10YJ25SW07)
)*+,

(u
(1986—),
?

ÈÉ/È1

ËE78&

78FG

H&œ

NÒ8

PQ1
),
RS
,Email:rbhuang@gxas.ac.cn
IJKHLMŠ%’;=>f?˜ŠN¿
§¨©
1,2,
Kˆ|
2,3,
ª
 
«
1,2,
K¬­
1,2,
®¯˜
1,2,
#°±
1,2,3
(1.
²³45 6eS58fg5<


530004;2.
²³S5< µ¢67¶·¸@ABC
DEF @Aµ¢67¶¹º2;fg»¼.l


530007;3.
012345 678
9:2;5<

01
210009)
>
 
?

±ˆ2‰2Š‹0Œ
(Kluyveromycesmarxianus)DSM5418
œ2Ž‡}
(INU)
Dª‘’“”‘’

e•–0Œ
(Pichiapastoris)GS115
œO€9%*p—Gr

Û@}˜™rs
1527U/mL,
“”•C‚©}“á9
:wäGš

Ê"
(NH4)2SO4›œ、Ëbp„"ž°,–s9:dK95%D:w‚©},SDSPAGEpË/
€
INU
DGŸRCp„ka«
90×104,
K@ >r¹
60×104。
:w}£DGšÇ!GH
,INU
DJâcd
bJâ
pH
pq«
55℃
b
50,
efVWg
INU
C‡D
Km¹b¤}˜pq«190mmol/Lb43386U/mg,C=
,D
Km¹b¤}˜pq«2781mmol/Lb124949U/mg,I/S¹«034;HPLCpËGH,INU}ݐ‡D2ç
¡!,bHI,©ª

¢£U„
Mn2+、Fe3+、K+
b
Co2+
C}í¤“Ð+

¥
Zn2+、Cu2+、Ni2+、SDS
b
EDTA
C}˜
™í:;dD)ÑÐ+

@AB

‡}

Gr

ˆ2‰2Š‹0Œ

•–0Œ
CDEFG
:Q5562    
HIJKL
:A    
HMNG
:1672-3678(2013)03-0040-06
Cloning,expressionandcharacterizationofexoinulinasefrom
KluyveromycesmarxianusDSM5418
XIONGWuping1,2,WANGChenghua2,3,LUYan1,2,WANGQingyan1,2,
SHENNaikun1,2,HUANGRibo1,2,3
(1ColegeofLifeScienceandTechnology,GuangxiUniversity,Nanning530004,China;
2NationalEngineeringResearchCenterforNonfoodBiorefinery,StateKeyLaboratoryofNonFood
BiomassandEnzymeTechnology,GuangxiAcademyofSciences,Nanning530007,China;
3ColegeofBiotechnologyandPharmaceuticalEngineering,NanjingUniversityofTechnology,Nanjing210009,China)
Abstract:ThematurepeptidegeneofexoinulinasefromKluyveromycesmarxianusDSM5418wascloned
intotheplasmidpPIC9K,whichwasfurtherexpressedeficiently,bysecretoryexpressedinPichiapastoris
GS115andmaximumactivityreached1527U/mL.Therecombinantinulinasewaspurifiedtobemore
than95% puritybyammoniumsulfateprecipitation,dialysisandsievechromatographyTheapparent
molecularweightofINUwas90×104bySDSPAGE,andwasbiggerthanthetheoreticalvalueof60×
104TheenzymaticcharacteristicsofINUwereasfolows:theoptimumtemperaturewas55℃ andthe
optimumpHwaspH50,theapparentKmandspecificactivityvaluesforinulinwere190mmol/Land
43386U/mg,and2781mmol/Land124949U/mgforsucrose,themetalionsincludingMn2+,Fe3+,
K+,andCo2+ activatedtheinulinaseactivity,butitwasinhibitedbyZn2+,Cu2+,Ni2+,SDS,and
EDTATheenzymatichydrolysisproductofinulinbyINUconsistedofglucoseandfructose,whichwas
consistentwiththeI/Svalueof034ofINU
Keywords:exoinulinase;expression;Kluyveromycesmarxianus;Pichiapastoris
  
0nIä78&éä

&Àä

&¡³
{’”h‡Ú&惷b)–ƒ·*÷hc!
±²ª“ðñ

#CSI%‚h‡Ú&æì·
?²78õö÷ôhHkOp‹!{

9@÷ôh
‡Ú&æì·M+k{SI%‚B˜
[1-2]。
+k
ÅcK¨¢@ø÷ߕ

‘,-@÷-* β 2,

÷þÿ÷¶

ƒ†àé.\Mc!õö÷ª
“

ø•\cûÅ
2~60。
+k€ñÂYóX|
Ðh+Ã

/—Ã{‹CX|Ðh.•Ã

0
øÃТ

1db+2O+3

d+k,Ñtˆ
•Òæ–
90%

[3],
b4Ø9èbh@÷ôá
ñ&ì·

á~‚Y&×&éä

3@÷j




ìN Àä

·¸W‹9Îä{׊
[4]。
¦Ú+kÎ
(EC32180)
 +k\h‡†àå
é.h÷þÿS…êcÚû@÷ô

e“áIJå

·
(D

),
b78Q+kÅàSIh‡Ú&
惷°ÿ"c

1¢#C ¶·[Öh“ü‹+
×&hÎlvíB]78

Áb÷°ôžh<þ‹3
}5=)+[ÖhʼFh78÷§B˜
[4-7]。
yz<þ½<5<6‹JÈ
DSM5418
¦Ú+
kΓ78
(INU)
hRWôž

ʼª“˜3}ñ
à5=
INU
h)+ˆ9:JÈ[Ö

Ölc–§±
ªÎlv׈OΎåæ78

Q#Å¡ãñX‚
{O)&‚§ôL

1 
OPQRS
11 
TUQŽ
½<5<6‹JÈ
(Kluyveromycesmarxianus)
DSM5418
è’_–H&[d„™¢š
(DSMZ);
ýeæÛ
pPIC9K,
<þ;?
EcoliXL10 gold
O5
=;?9:JÈ
(Pichiapastoris)GS115
Jè’
Invitrogen
ïð

ք™Y«ÃŽÄ&¢š[d
‘

JÈækgõö÷òóô
(YEPD)
‰4(
[8],
‚ßõö÷òóô
(MD)、
ôøòóô
(MM)、
‚ß%&9³j•òóô
(BMGY)
O‚ß%&àä
j•òóô
(BMMY)
‰3
Invitrogene
ïðh9:J
È5=Š&

+kèY
Sigma
ïð

ÊËå”ÚÎ
O
T4DNA
¶MÎè’
MBI
ïð

æÛp¤
、PCR
×
׈OpUÓ¤TIANGEN
ïð

SephacrylS 300
è’
GE
ïð

dšˆŽ¤Jè
’–‡Ô

[/ôÿZKôžÏÿX‡=
½1&)#“

12 
RS
121 
+kÎôžh<þ
Q½<5<6‹JÈ
DSM5418
>
DNA
Å 
µ

ñ­Q
P15′TGACCTAGGATGATGGATGGT
GACAGC 3′
O
P25′ATAGCGGCCGCATTAAAG
GTTAAATT 3′
ŽûÐ[
,PCR
ãu+kΓ
78ôž

2PÅF’ôž«5

ñ­’½Ð[
P1
¢[ãÊËå”ÚÎ
AvrI
ÎÚ9é

û<#ð
ñ
),
’ûÐ[
P2
¢[ã
NotI
ÊËå”ÚÎÎ
Ú9é

û<#ðñ
)。
122 
±ªæÛhʼ

̈‹“üX
PCR
×OæÛ
pPIC9K
hÎÚ

pUÓ

¶M
Ö«5‰3(
[8]
lv


,PCR
×O
æÛ
pPIC9K
ñ­‚
AvrI
O
NotI
óÎÚ

¶M×
̈ž
EcoliXL10 gold
ÏÇب©

±ªæÛ
-*:9h1!yå“ü

ÎڐXO
DNA
Ïÿ
X

Xí€h±ªæ۟1Å
pP9I。

pP9I
‚
SacI
CÎÚ#刡

$̈
PichiapastorisGS115
ÏÇب©

̈¨©>ÂY
MD
uµ½
,30℃
ò
ó
2~3d。
N¤C[Šñ­é’
MM
O
MD

½
,30℃
òó
2~3d,
’
MM
O
MD
½J&[íA
h[ÖdÅ
Mut+
̈X


Mut+
̈Xédž
Â^2‡\h
G418
uµ½

“ü]?@±ªX

Ö
Xí€h±ª[ÖAäÅ9:JÈ
P9I。
123 
)+[hçp5=O±ªÎh׈
9:JÈ
P9I
Mdž
BMGY
òóô¢
,30℃、
250r/min´
µòó¯
OD600=2~6,WšÓÔ[e,
‚
BMMY
±ÿ¨©¯
OD600=10,’ 30℃、250
r/min
û6ãòólvçp5=

¥Œ
24h
¤½«
ÏάÖÆÀ(à䯏æ–ñŠÅ
05%
Q6ã
çp
96h。
Y
4℃、3500r/min
ÌÍûWšÓÔç
14 
!

# (u{

c!¦Ú+kÎôžh<þ5=‹ÎŽåæ
pò󿽫

½«¢±ªÎ-*Qû–Blv×
ˆ

!c–

½«¿¢(ã
(NH4)2SO4GH¯_O

40%
Q®¯kÀf

ñ(ã
(NH4)2SO4¯_O

80%
QGH6hÀf

!Á–

GH±ÿY÷
e_h
02mol/L
CW%&r¿
(pH50)
¡

>
ýYC‹–Å
10×104
hXíD¢

Y2óh%&
¿¢Xí
24h,
¢wiL%&¿

a

¢¡‚
PEG
20000
‡Ò¯
4mL;
!1–

EõY
AKTA
Àf׈



4mL
‡Ò¤ó-*
SephacrylS300
ñX“À
íñW

d¢üXŸÍÅ
25cm×80cm,
«5þÍÅ
1mL/min,
þ¸÷£‚
02mol/L
CW%&¿
(pH
50),
‰
45mL
e_ñvÓÔʳ¿

׈Àfh
×\OJcå-*
SDSPAGE
$Ü[

Àfh–

Bradford

[9],
Q
BSA
ÅÐÑÀflv[Ï

124 
άhρ
+kÎάhρ‰|
Pessoni
{
[10]
hFÖ
ñ5F‚

¤
1mL
†PNOh×ο(ã
4mL20
g/L
+kÙ:÷
(02mol/L、pH50
CW%&¿/
Ë
),
Y
55℃
tå¢.
10min,
¤
05mL
.¿

15mL
tO
15mL3,5
ÁGôt¹W
(DNS),
Y¥tå¢
5min,
ÆðÍçè¡Y
540nm
Kρ&“h†à÷–

Q’¥tå¢(¦Å¬
hο5ŧ3

Q+kO:÷Å+Pρ
hάñ­Ÿ1Å

O
S。
܁x

’
55℃、pH50(02mol/L
CW%
&¿

ôõû

¥ñ;×&
1μmol†à÷+ghÎ
–

ÇÅ

!άnC9
(U)。
125 
Ύåæhρ
1)
‚†ˆ\O¦Ýåhρ
 
‰3
124
Î
¬ÏF

ñ­’
30~80℃
ûρά

Qρ
3@‚3άÅ
100%,
;0mρªh÷§Î¬

†PNOhο’
30~80℃
û„ˆ
30min,
ðÍç
è

ϙšÎ¬

Q?C*KLÎhάn
Å
100%。
2)
‚†
pH
O
pH
݁åhρ
 
‰3
124
άρF

’
02mol/L、pH30~70
CW%
&¿ûρά

Q‚3άÅ
100%,
;0mρ
ªh÷§Î¬

ο‚^2‡pH
%&¿NO¡

4℃
„ˆ
1h,
ϙšÎ¬

Q?C*KLÎhά

100%。
3)
VXWX
、SDS
‹
EDTA
§ÎhST
 

k.eQ¢(ã^2hVXWX
、SDS
‹
EDTA,
>d‡\Å
05mmol/L,
Q?KLÎάnÅ
100%,
;0(ã^2æ¡h™šÎ¬

4)KmOO Vmaxhρ £‚ LineweaverBurk
óHŠ56¶Ï

ñ­Q+kO:÷Å+
ρS,’55℃、pH50ôõû,.5min,ρ’C9
Pw”†à÷hu(–

;0.„Ív。
Q
1/v
O
1/ρS56֕KmOOVmaxO。
126 HLPC
ñítÏ×
‰3
1∶20


¤*–h×Îñ­K
20g/L
+kO
20g/L
:÷’
50℃、pH50(
CW%&¿

ôõû.
16h
QI+tÏ+

¢¡’¥tå¢
ù.

ÎÏ×C
12000r/min
WšO
022
μm§Œ*§¡lvHPLCñí。
2 
_`Qab
21 
LMŠ%’;=>
Q½<5<6‹JÈôžªÅ µ

Q
P1
O
P2
Å[
,PCR
ãu•žÄÅ
16kb
h6hv
(GenBank
JKSÅ
JQ411233)。
ÿZñí5;

“
78+kÎ
INU
hL§MÌÅ
1602bp,
RW
534
:ôW

œ”ÂX

ij

!–’h÷ôˆ9é

Blast
ñí3@5;
,GenBank
¢Åj

!#ˆh<
6‹JÈ+kÎôž
,INU
K78
EU716512、
X572021、AF1355941、X684791、AB6215731
O
AF1789791
’”h

!ôžij3=
99%
h2·
å

K
AY6494431
h2ጁ
95%。
22 
‘l¿5Åg¥T;OP
PCR
×O5=ýe
pPIC9K
CóÎÚ

¶M

ʼ5=æÛ
pP9I。
¸å<þp¤æÛ¡

C
AvrI
O
NotI
óÎڐX

ÎÚ3@
ôx

Kýe
O6hôž0ß÷ѕ

6
1),
Ïÿ3@5;S"L
§M
(ORF)
í€

žŸ›;

±ªæÛʼ“|

M—λ/HindIIDNAÐó;1—pPC9KNotICÎÚ;
2—pP9I
±ªæÛ
AvrI
O
NotI
óÎÚ
D
1 
f?¿5
pPIC9K INU
;ŠH^4
Fig.1 VerificationofexpressionplasmidpPIC9KINU
24
&
 

 
(
 
)
 
*
 
+
  
!
11
"
 
pP9I
C
SacI
ÎÚ#刡

$̈l9:JȨ
©

C
MD、MM
‹^2‡\h
G418
“ü

‚•ž

Ö
His+/Mut+
3?@h¸å̈X

C
PCR
X
,INU
ôž“|ˆ•l9:JÈôžª¢

üýd¢

Ö)
+[Ö

Ð1Å9:JÈ
P9I,
‚Y¡ã׈)5

23 
g¥T;ÊËf?˜ŠàŒ
‰3F
123
+/
,BMMY
òóôòóh9
:JÈ
P9I
’
05% (
e_ñŠ

àäçphŽ
û

•ží3}ñà5=

C*
(NH4)2SO4GH、X
íOñX“Àí

׈•ží$Ü×h±ªÎ

5
1)。SDSPAGE
[Ï5;
,INU
h5`÷§ñXæ–
ÄÅ
90×104,
0YLY÷§ñXæ–
60×104

6
2)。
e K
Zhang
{
[11]
× ˆ
Kluyveromyces
marxianus
¦Ú+kÎÅ
85×104,
LYOÅ
62×
104
^2

9K
Lim
{
[12]
78h+kÎ5`÷§ñ
Xæ–Å
98×104
h3@@£

f
1 
‘lLMŠ;àŒÃ¥
Table1 SummaryofpurificationofinulinasefromPichiapastorisP9I
«5–B
>¬n


m(
Àf
)/
mg
gά

(U·mg-1) ׈æŠ
UÓ*


à…IJ¿
1328845 31821 4176 0 100
(NH4)2SO4GH 882900 13728 6431 154 6644
Xí³F
620298 7176 8644 207 4668
ñX“Àí
169639 391 43386 1039 1277
M—
ÀfæÐÑ
;1—
±ªæÛ
pP9I
h)+ˆ
9:JÈ
P9I;2—
Â2æÛ
pPIC9K
h9:JÈ
GS115;
3~6—
׈h±ª+kÎ
INU
D
2 
‘lŠàŒè;
SDSPAGE

Fig.2 SDSPAGEanalysisofpurifiedINU
fromPichiapastorisP9I
24 
ŠN¿¼+
241 
‚†.ˆ\‹¦Ýå
¦Ú+kÎ.h‚†ˆ\‹¦Ýåh7
83@46
3。
‘6

á7

:Îh‚†ˆ\Å
55℃,
’
35~60℃
"wάnƒž=
60%


‚
†ˆ\K
Zhang
{
[11]
O
Sarup
{
[6]
•žh<6‹J
ȦÚ+kÎcq

Î¿’^2ˆ\û„ˆ
30
min
ρN¦å

3@55℃
Qû„ˆ
30min
ij
80%
Q½h¬n



:Îijhª€5‚
ˆ\‹B3hˆ\NÇå

j%Y&+krÏK
d:)h*+MDEIJ*+{P•>‚

D
3 
zscŠªNÅQ+N;de
Fig.3 Efectsoftemperatureonactivityand
stabilityofinulinase
242 
‚†
pH
‹
pH
݁å
¦Ú+k΂†.
pH
‹
pH
݁åh3@
46
4。
‘6

á7

:Îh‚†
pH
Å
50,
’
pH
30~60
Îij
50%
Q½h¬n
,pH
()B€

eK#)*hJÈX+k΂†
pH
’
44~65[6]
ôøcq

’^2
pH
¢
4℃
‰å„ˆ
1h
¡

3@
I3’
pH40~65
B݁

áQ„â0Ä
90%
h
÷§¬n

:ÎhW傆
pH
‹d’ª€hWå
pH
()”h3\NÇå

j%Y+khtÏ

Öá
Own!O6×he“

243 
VXWX
、SDS
O
EDTA
§ÎhST
VXWX
、SDS
O
EDTA
§¦Ú+kÎhS
T3@46
5。
‘6

á7
:Mn2+
§:ÎjBv
34 
!

# (u{

c!¦Ú+kÎôžh<þ5=‹ÎŽåæ
hkl5‚
,Fe3+、K+
O
Co2+
§Î_j^2+hkl5‚

9
Zn2+、Cu2+、SDS、Ni2+
O
EDTA
§
άnjBvhcË5‚
,Mg2+
§ÎhST^
;<

D
4 pH
cŠªNÅQ+N;de
Fig.4 EfectsofpHonactivityand
stabilityofinulinase
244 KmO、VmaxO‹I/SOhρ
’‚†
pH50
O‚†ˆ55℃
ôõû

£‚
LineweaverBurk
óHŠ56

ρ•ží
INU
%
‚+khΎ‰Š

2P

Åíρ
INU
h
I/S
O

’÷2hρôõû

yzρí
INU
Q:ց+
     
D
5 
&Rü“
、SDS
Å
EDTA
c‘lŠ;de
Fig.5 Efectsofmetalions,SDS,andEDTA
oninulinaseactivity
PhΎ‰Š

5
2)。
 5

á4
,INU
%‚+
kh‚0.Í*Å
1171mmol/(L·min),
5`i
ÝAŠÅ
190mmol/L,
9%‚:÷h‚0.Í

2899mmol/(L·min),
5`iÝAŠÅ
2781
mmol/L,
á4
INU
h‚†¨¢+Å+k
。INU
%
‚+kO:÷he_άnñ­Å
2132U/mL
O
614U/mL,
9g¬nñ­Å
43386U/mg
O
124949U/mg,
Yz"gÅ
034,
ÇÅ
I/S
O
,I/S<
10,
5;:ÎŦÚ+kÎ
[13-14]。
f
2 
LMŠ;
KmS、VmaxS˜I/SS
Table2 Km,VmaxandI/SratioofinulinasefromKmarxianus
+
Km/
(mmoL·L-1)
Vmax/
(mmoL·L-1·min-1)
e_ά

(U·mL-1)
gά

(U·mg-1)
I/S
+k
190 1171 407 43386 034

2781 2899 1209 124949
  
Ÿ3@K
Kushi
{
[7]
)*h+kΧ+k
Km=
869mg/mL、

Km=458mg/mLh3@^
2
。INU
§+kh3+©OnOŒˆ¬å

i(
j%Ys‡\ôõû+khëñ%‚

jõYrs
)&&×*+¢+kh+™‹

245 
ÎtÏ+kO:÷×ñí
Ålc–X
INU
h¦Úάå

ñ­¤*–
h×Î>+kO:÷I+tÏ

‚
HPLC
ñítÏ
×

3@46
6。
‘6

á7

+kÎÏ¿¢?²
“ñÅ@÷

†j˜–hõö÷

:÷ÎÏ¿¢?
²Å@÷Oõö÷

Áz–ôø÷2

3@
lc–5;
,INU
ŦÚ+kÎ

áQ+krϔ
•3×\h@÷‹õö÷

 9ŇÚ&惷
{&×*+páIJå

·

D
6 
LMŠÚµLMÅ01
HLPC
ÒDD
Fig.6 HPLCchromatogramoftheproductsfrom
inulinandsucrosebyinulinase
44
&
 

 
(
 
)
 
*
 
+
  
!
11
"
 
3 
_
 
b
“|<þí½<5<6‹JÈ
DSM5418

78¦Ú+kÎ
(INU)
ôž

-*ʼ±ªPQæ
Û
pP9I,
“|ʼí3}ñà5=±ª
INU
h)+
ˆ9:JÈ
P9I。

(NH4)2SO4GH、Xí‹
SephacrylS 300
¥pñX“F÷3•”•í$
Ü×h±ªÎ

±ªÎ
INU
h5`÷§ñXæ–
Å
90×104,
‚†ˆ\O‚†
pH
ñ­Å
55℃
O
50,I/S

034,
53ŦÚάå

ôY+k
O:÷‚LM×h
HPLC
ñí3@lc–X
í¦Úάå
。Mn2+、Fe3+、K+
O
Co2+
§Îjkl


9
Zn2+、Cu2+、SDS、Ni2+
O
EDTA
§Î¬nj
^2+\hcË5‚

ø78Å
INU
h¡ãG
lˆ)5‹lc–SäôY+2h‡Ú&æì
·%‚§íôL

‚ƒHI

[1] 
yî_

+2Ëî&éäh78lä
[J].
ÈÉâ&Î

2010,38(26):1481914820.
[2] 


,ƒN

ySv

{

TUüŒ<6‹JÈ%‚+2à
2–÷ˆKIJ&×éä
[J].
&)+Ž)
,2010,26
(7):982990.
[3] 
m¼Z

H&+kÎôž3Ê

ΎåæK‚78lä
[J].¨
¢×78KSI
,2001,13(1):8389.
[4] PandeyA,SoccolCR,SelvakumarP,etal.Recentdevelopments
inmicrobialinulinases[J].ApplBiochemBiotechnol,1999,81
(1):3552.
[5] SinghRS,DhaliwalR,PuriM.Productionofhighfructosesyrup
from Asparagusinulin usingimmobilized exoinulinasefrom
KluyveromycesmarxianusYS1[J].JIndMicrobiolBiotechnol,
2007,34(10):649655.
[6] SarupSRAM,DhaliwalR,PuriM.Partialpurificationand
characterizationofexoinulinasefrom Kluyveromycesmarxianus
YS1forpreparationofhighfructosesyrup[J].JMicrobiol
Biotechnol,2007,17(5):733738.
[7] KushiRT,MontiR,ContieroJ.Production,purificationand
characterizationofanextracelularinulinasefromKluyveromyces
marxianusvar.bulgaricus[J].JIndMicrobiolBiotechnol,2000,
25(2):6369.
[8] 
VWÂ6<
J,
X®“
DW.
ñX<þnA
[M].3
„

ö


Î愘
,2002.
[9] BradfordMM.Arapidandsensitivemethodforthequantitation
ofmicrogram quantitiesofproteinutilizingtheprincipleof
proteindyebinding[J].AnalBiochem,1976,72(1):248254.
[10] PessoniR,FigueiredoRibeiroR,BragaM R.Extracelular
inulinasesfromPeniciliumjanczewski,afungusisolatedfromthe
rhizosphereofVernonia herbacea(Asteraceae)[J].JAppl
Microbiol,1999,87(1):141147.
[11] ZhangL,ZhaoC,OhtaW Y,etal.Inhibitionofglucoseonan
exoinulinasefromKluyveromycesmarxianusexpressedinPichia
pastoris[J].ProcessBiochem,2005,40(5):15411545.
[12] LimSH,LeeH,SokDE,etal.RecombinantProductionofan
inulinaseinaSaccharomycescerevisiaegal80strain[J].J
MicrobiolBiotechnol,2010,20(11):15291533.
[13] NakamuraT,ShitaraA,MatsudaS,etal.Production,purification
andpropertiesofanendoinulinaseofPeniciliumsp.TN88that
liberatesinulotriose[J].JFermentBioeng,1997,84(4):
313318.
[14] ParkJP,KimDH,KimDS,etal.Enzymaticproductionof
inulooligosaccharidesfromchicoryjuice[J].BiotechnolLet,
1998,20(4):385388.
54 
!

# (u{

c!¦Ú+kÎôžh<þ5=‹ÎŽåæ